Enhancing Reactivation of Thermostable Reversibly Inactivated Enzymes

ABSTRACT

Disclosed are methods for the enhancement of the reactivation of thermostable reversibly inactivated enzymes comprising reactivating at least one thermostable reversibly inactivated enzyme in the presence of one or more nitrogen containing compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. patentapplication Ser. No. 11/821,173, filed Jun. 22, 2007, which claimsbenefit to U.S. Provisional Application No. 60/817,043, filed Jun. 28,2006, and 60/852,804, filed Oct. 19, 2006, all of which are incorporatedby reference in their entireties.

BACKGROUND

Some molecular biology methods demand enzymes that can be activated in acontrolled manner while being already contained in the reaction. Thisallows starting sequential enzymatic procedures in the same reactioncontainer at pre-defined time points or avoiding the generation ofundesired side products due to premature start of the enzymaticreaction. For the use of chemically modified thermostable enzymes, twosuch modifications have been described for thermostable DNA polymerases(see U.S. Pat. Nos. 5,677,152; 5,773,258; and 6,183,998, which areincorporated by reference herein in their entireties). Suchmodifications can also be employed for other thermostable enzymes sinceamino acids become modified by these chemicals that are contained in allenzymes and are often involved in the catalytic centre of enzymes suchas amino acids carrying a free NH₂-group. Furthermore, U.S. Pat. No.6,183,998 teaches the crosslinking of enzyme molecules by aldehydes,which is generically suitable to modify any thermostable enzyme. Theactivity of such chemically modified enzymes becomes restored by a heatincubation step that breaks the covalent bonds to the amino acidresidues thereby restoring the catalytic activity of the enzyme.However, depending on the inactivation procedure or the modifierconcentration chosen, enzyme activity is restored only at a slow rate.

The inventors have discovered that nitrogen-containing compoundssurprisingly enhance enzyme activity of chemically modified enzymeswhile they have no effect on the unmodified enzyme. Thus, this inventiondescribes for the first time a mechanism that enhances the reactivationof chemically modified enzymes, which is suitable in many applications,e.g., to increase speed of the enzymatic reaction.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, articles, and methods, as embodied and broadly describedherein, the disclosed subject matter, in one aspect, relates tocompounds and compositions and methods for preparing and using suchcompounds and compositions. In a further aspect, the disclosed subjectmatter relates to the use of one or more nitrogen containing compoundsto enhance reactivation of reversibly inactivated thermostable enzymes.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an agarose gel from a fast PCR reaction with varyingconcentrations of Triazole.

FIG. 2 is a group of tables and accompanying graphs, each table showingaverage C_(T) values from reactions with varying concentrations of aparticular ammonia compound.

FIG. 3 is a group of three agarose gels from three fast PCR reactionswith varying concentrations of ammonium chloride.

FIG. 4 is an agarose gel from a fast PCR reaction in the presence ofammonium chloride and varying concentrations of Triazole.

DETAILED DESCRIPTION

The materials, compounds, compositions, articles, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subject matterand the Examples included therein and to the Figures.

Before the present materials, compounds, compositions, articles, andmethods are disclosed and described, it is to be understood that theaspects described below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes mixtures of two or more such compounds, reference to “anenzyme” includes mixtures of two or more such enzymes, reference to “theprimer” includes mixtures of two or more such primers, and the like.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can also be substituted or unsubstituted. The alkyl groupcan be substituted with one or more groups including, but not limitedto, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, boronic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, hydroxamate, silyl, sulfo-oxo, or thiol, as describedherein. A “lower alkyl” group is an alkyl group containing from one tosix carbon atoms.

The term “alkoxy” as used herein is an alkyl or cycloalkyl group bondedthrough an ether linkage; that is, an “alkoxy” group can be defined as—OA¹ where A¹ is alkyl or cycloalkyl as defined above.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This may be presumedin structural formulae herein wherein an asymmetric alkene is present,or it may be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, boronic acid, ester, ether, halide,hydroxy, ketone, azide, nitro, hydroxamate, silyl, sulfo-oxo, or thiol,as described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, boronic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, hydroxamate, silyl, sulfo-oxo, or thiol, as describedherein.

The term “acyl” means derivatives of the corresponding acids comprising10 or less continuous C-atoms. These acid moieties can carry additionalalkyl groups or functional groups like hydroxyl, amino, carboxyl,sulfonic acid, ester or ether groups.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, boronic acid, ester, ether, halide,hydroxy, ketone, azide, nitro, hydroxamate, silyl, sulfo-oxo, or thiolas described herein. The term “biaryl” is a specific type of aryl groupand is included in the definition of “aryl.” Biaryl refers to two arylgroups that are bound together via a fused ring structure, as innaphthalene, or are attached via one or more carbon-carbon bonds, as inbiphenyl. The term “phenyl” is included within the meaning of aryl andmeans a benzyl moiety, which can carry additional alkyl chains(comprising 4 or less continuous C-atoms) or functional groups likehydroxyl, amino, nitro, carboxyl, sulfonic acid, esters, ether orhalogen groups, attached to the ring.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen orsubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “amino alkyl” means an amino group with an alkyl moiety. Theterm “alkyl” is defined above. Equally, derivatives of amino alkylcorrespond to the derivatives of alkyl groups.

The term “azole” as used herein is a substituted or unsubstituted orfused five-membered ring containing at least one nitrogen atom in thering.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, a substituted or unsubstitutedalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein.

The term “halide” or “halogen” as used herein refers to the halogensfluorine, chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, a substituted or unsubstitutedalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or a substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen ora substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Throughout this specification “S(O)” is a short hand notation for S═O.The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂A¹ , where A¹ can be hydrogen or asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “sulfone” as used herein is represented by the formulaA¹S(O)₂A¹, where A¹ and A¹ can be, independently, a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein. The term“sulfoxide” as used herein is represented by the formula A¹S(O)A¹, whereA¹ and A¹ can be, independently, a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” and “X,” as used herein can, independently, possess one ormore of the groups listed above. For example, if R¹ is an alkyl group,one of the hydrogen atoms of the alkyl can optionally be substitutedwith a hydroxyl group, an alkoxy group, an alkyl group, a halide, andthe like. Depending upon the groups that are selected, a first group canbe incorporated within second group or, alternatively, the first groupcan be pendant (i.e., attached) to the second group. For example, withthe phrase “alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Methods and Compositions

The present invention provides a method to enhance reactivation ofreversibly inactivated thermostable enzymes. In particular, thedisclosed methods involve the use of nitrogen containing compoundscharacterized by the following Formula 1-6

where each X is, independent of the others, CH, CR, N, O, or S; and eachR is, independent of the others, H, substituted or unsubstituted alkyl,alkenyl, alkynyl, alkoxyl, or aryl, a hydroxyl, amino, nitro, carboxylicacid, ester, sulfonic acid, ether, or halogen group. As disclosedherein, these compounds, including mixtures thereof, can be employed toenhance reactivation of thermostable reversibly inactivated enzymes.

In various examples, disclosed herein are methods for the enhancement ofthe reactivation of thermostable reversibly inactivated enzymes thatcomprise reactivating at least one thermostable reversibly inactivatedenzyme in the presence of a nitrogen-containing compound. Specificexamples of nitrogen containing compounds suitable for use hereininclude, but are not limited to, an azole. Suitable azoles can comprisea nitrogen-containing 5-membered cyclic compound having Formula 1,

where each X can be, independent of the others, CH, CR, or N; and R canbe H, substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, oraryl group, a hydroxyl, amino, nitro, carboxylic acid, ester, sulfonicacid, ether, or halogen group.

In another example, a suitable azole can comprise a nitrogen-containing5-membered cyclic compound having Formula 2,

where each X can be, independent of the others, CH, CR, N, O, or S; andeach R can be, independent of the others, H, substituted orunsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl group, ahydroxyl, amino, nitro, carboxylic acid, ester, sulfonic acid, ether, orhalogen group. In a further example, each X can be, independent of theothers, CH or N in any combination.

In yet a further example, a suitable azole can comprise anitrogen-containing bicyclic compound having Formula 3,

where each X can be, independent of the others, CH, CR, N, O, or S; andeach R can be independent of the others, H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, alkoxyl, or aryl group, a hydroxyl, amino,nitro, carboxylic acid, ester, sulfonic acid, ether, or halogen group.

Also disclosed herein are methods for the enhancement of thereactivation of thermostable reversibly inactivated enzymes thatcomprise reactivating at least one thermostable reversibly inactivatedenzyme in the presence of a compound comprising a pyridine havingFormula 4,

where each X can be, independent of the others, CH, CR, or N; and R canbe a hydroxyl, amino, thiol, or amino alkyl group, or derivativesthereof.

Still further, disclosed herein are methods for the enhancement of thereactivation of thermostable reversibly inactivated enzymes thatcomprise reactivating at least one thermostable reversibly inactivatedenzyme in the presence of a compound comprising an N-hydroxide havingFormula 5,

where each R can be, independent of the other, H, substituted orunsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl group, or anacyl group or derivatives thereof.

In yet a further example, disclosed herein are methods for theenhancement of the reactivation of thermostable reversibly inactivatedenzymes that comprise reactivating at least one thermostable reversiblyinactivated enzyme in the presence of a compound comprising a hydrazinehaving Formula 6,

where each R can be, independent of the other, H, substituted orunsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl group, or anacyl group or derivatives thereof.

In another example, disclosed herein are methods for the enhancement ofthe reactivation of thermostable reversibly inactivated enzymes thatcomprise reactivating at least one thermostable reversibly inactivatedenzyme in the presence of an ammonium salt. An ammonium salt is anycompound that contains a positively charged nitrogen species of theformula ′NR₄, where each R can be, independent of the others, H,substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl,a hydroxyl, amino, nitro, carboxylic acid, ester, sulfonic acid, ether,or halogen group, and a negatively charged inorganic counterion, organiccounterion, a metal oxide counterion, or a metal complex counterioncounterion.

Suitable positively charged nitrogen species for the ammonium salts areNH₄ and tetraalkyl ammonium. Suitable counterions in the ammonium saltscan have one or more negative charges. Examples of suitable counterionsinclude, but are not limited to, inorganic counterions like halogens(e.g., F⁻, Cl⁻, Br⁻, and I⁻), pseudohalogenes (e.g., SCN⁻ and OCN⁻),perchlorate (ClO₄ ⁻), chlorate (ClO₃ ⁻), chlorite (ClO₂ ⁻), hypochlorite(ClO⁻), bromate (BrO₃ ⁻), periodate (IO₄ ⁻), sulfide (S²⁻), sulfate (SO₄²⁻), sulfite (SO₃ ²⁻), bisulfate (HSO₄ ⁻), phosphide (P³⁻), phosphate(PO₄ ³⁻), hydrogen phosphate (HPO₄ ²⁻), dihydrogen phosphate (H₂PO₄ ⁻),phosphite (PO₃ ²⁻), nitride (N³⁻), nitrate (NO₃ ⁻), nitrite (NO²⁻),hydroxide (OH⁻), peroxide (O₂ ²⁻), hexaflourophosphate (PF₆ ⁻), andpermanganate (MnO₄ ⁻).

Further examples of suitable counterions include, but are not limitedto, organic counterions like carbonate (CO₃ ²⁻), bicarbonate (HCO₃ ⁻),formate (CHO₂ ⁻), acetate (CH₃CO₂ ⁻) and other alkyl carboxylates (RCO₂⁻, where R is alkyl, e.g., propionate, buterate, etc.), oxylate (C₂O₄²⁻), tartrate and malate (C₄H₄O₆ ²⁻), citrate (C₆H_(S)O₇ ³⁻), andbenzoate (C₆H₅CO₂ ⁻).

Other suitable counterions include, but are not limited to, metal oxidesand metal complexes. Examples of metal oxides and metal complexesinclude orthovanadate, molybdate (Mo₇O₂₄), hexacyanoferrate (FeCN₆)⁴⁻,and cerium nitrate.

Any of the counterions disclosed herein can be combined with any of theammonium species disclosed herein to form an ammonium salt. Somespecific examples of suitable ammonium salts include, but are notlimited to, ammonium chloride (NH₄Cl), ammonium dihydrogen phosphate(NH₄H₂PO₄), ammonium biphosphate ((NH₄)₂HPO₄), (NH₄)₃PO₄,(NH₄)₄(Fe(CN)₆), ammonium acetate (NH₄CH₃CO₂), ammonium carbonate(NH₄)₂CO₃), ammonium molybdate (NH₄Mo₇O₂₄), ammonium perchlorate(NH₄ClO₄), ammonium citrate ((NH₄)₃C₆H_(S)O₇), ammonium benzoate(NH₄C₆H₅CO₂), and ammonium phosphate ((NH₄)₃PO₄).

It is understood that some adjustments can be made to the methodsdisclosed herein. For example, if a counterion of the ammonium saltchelates Mg from the reaction, which is required by the enzyme as aco-factor, the salt can still provide the same effect on reactivation.However, one would have to increase the Mg concentration of the reactionto compensate for the chelating effect of the counterion. Also, if acounterion has an inhibitory effect on the enzyme, one can optimize fora concentration that is effective but still not inhibitory.

In one particular aspect, the ammonium salt can be used with any of theazoles, pyrimidines, N-hydroxylamines, or hydrazines disclosed herein.As is discussed more fully below, the use of an ammonium salt, asdisclosed herein, with any of the other nitrogen containing compoundsdisclosed herein can result in a synergistic effect.

In the methods disclosed herein, the azoles, pyrimidines, N-hydroxides,and hydrazines, disclosed herein can be present at a concentration offrom about 0.5 mM to about 1 M, e.g., about 0.5, 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320,325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460,465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530,535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600,605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740,745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880,885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950,955, 960, 965, 970, 975, 980, 985, 990, 995, 1000 mM, where any of thestated values can form an upper or lower endpoint of a range. For theammonium salts, the ammonium species can be present at any of theconcentrations just listed.

In certain examples, the ammonium compound is not ammonium citrate,di-ammonium hydrogen phosphate, ammonium benzoate, ammonium carbonate,or ammonium sulfate.

The disclosed methods can be employed in all applications involvingmanipulation of nucleic acids, such as amplification, ligation,exonucleolytic or endonucleolytic reactions, or nucleic acid topologychanging enzymatic reactions, wherein the inactivated enzymes becomereactivated by incubating the reaction mixture prior or as part of theintended enzymatic reaction at an elevated temperature in the presenceof nitrogen-containing compounds according to the description.

EXAMPLES

The following examples are set forth below to illustrate the methods andresults according to the disclosed subject matter. These examples arenot intended to be inclusive of all aspects of the subject matterdisclosed herein, but rather to illustrate representative methods andresults. These examples are not intended to exclude equivalents andvariations of the present invention which are apparent to one skilled inthe art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, desired solvents,solvent mixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

As model system to exemplify the invention, hot start PCR was chosenwith two commercially available enzymes. This shows the genericsuitability of the invention because both polymerases were inactivatedwith a different chemical modifier. AmpliTaq Gold (Applied Biosystems)is chemically inactivated using dicarboxylic acid anhydride whereasHotStarTaq (QIAGEN) is chemically inactivated using formaldehyde. Hotstart PCR was chosen as model system for the following reasons: (i) Thepolymerase chain reaction is a widely used molecular biology applicationand thus illustrates the economical importance of the invention, (ii)due to the inherent high efficiency of the reaction, PCR is a good modelsystem to show that the substances used in the invention do notinfluence reactions that contain an unmodified enzyme and secondly caneven further enhance the efficiency of such reaction even further if achemically modified enzyme is contained.

To easily demonstrate the increase of enzyme reactivation efficiency,real-time PCR was chosen. In real-time PCR, the enzymatic reaction iscontinuously monitored by measuring fluorescence signals throughout thereaction. Double-stranded DNA molecules are generated during the PCRreaction. These PCR products are detected by using SYBR Green Ifluorescent dye that binds selectively to dsDNA only. Thus, an increaseof fluorescence indicates an increase in PCR product amount and thusactivity of the DNA polymerase contained in the reaction. Once the totalfluorescence significantly increases above the fluorescent backgroundsignal, the so-called C_(T) value is reached. Thus, the C_(T) value is ameasurement for the enzyme activity contained in the reaction: Low C_(T)values indicate high enzyme activity and high C_(T) values representlower enzyme activity residing in the reaction.

Yet, another example shows a titration of one of the chosen substances(Triazole) and its effect on increasing the enzyme activity in PCR bythe generation of concentration dependent PCR product yields.

Example 1

A SYBR Green I based PCR reaction was conducted in the presence ofunmodified Taq DNA Polymerase (QIAGEN) serving as a control reaction,using HotStarTaq DNA polymerase (QIAGEN) or AmpliTaq Gold (AppliedBiosystems). Each reaction contained 0.625 units of the respective DNApolymerase, 1× PCR reaction buffer of the respective DNA polymerase, 200μM of each dNTP, 0.5 μM primer A: 5′-GTC ACC TTC ACC GTT CCA GT-3′ (SEQID NO:1), 0.5 μM primer B: 5′-CTC TTC CAG CCT TCC TTC CT-3′ (SEQ IDNO:2), PCR grade water and in case of testing nitrogen-containingcompounds as example 100 mM Triazole, final concentration. Each reactioncontained 10 ng human genomic DNA as amplification substrate. PCRreactions were started with an initial enzyme activation step at 95° C.for 5 minutes followed by 50 cycles: 94° C. for 15 seconds and 60° C.for 60 seconds. Average C_(T) values were determined from triplicatereactions comparing reactions without the addition of Triazole and with100 mM Triazole.

Average C_(T) value Average C_(T) value with Enzyme without Triazole 100mM Triazole Taq (unmodified) 21.0 21.3 HotStarTaq 42.3 35.9 AmpliTaqGold 27.6 25.6 Result: While the addition of Triazole showed no effecton the enzyme activity when using unmodified Taq DNA polymerase,reactions containing Triazole showed clearly higher enzyme activity whenusing both chemically modified enzymes, HotStarTaq DNA Polymerase andAmpliTaq Gold.

Example 2

A fast PCR reaction with significantly shortened cycling times wasconducted containing the chemically modified HotStarTaq DNA Polymerasein the absence of Triazole and in the presence of increasing Triazoleconcentrations. Each PCR reaction contained 1.5 units HotStarTaq DNAPolymerase, 10 ng template human genomic DNA, 200 μM of each dNTP, 0.5μM primer A: 5′-CAC ACA GCG ATG GCA GCT ATG C-3′ (SEQ ID NO:3), 0.5 ′Mprimer B: 5′-CCC AGT GAT GGG CCA GCT C-3′ (SEQ ID NO:4), PCR grade waterand in case of testing nitrogen-containing compounds as example 100 mM,200 mM, 300 mM, 400 mM and 500 mM Triazole, final concentration. PCRreactions were started with an initial enzyme activation step at 95° C.for 5 minutes followed by 35 cycles: 96° C. for 5 seconds, 60° C. for 5seconds and 68° C. for 30 seconds. PCR products were analysed on a 1%TAE agarose gel (FIG. 1).

Result: The fast PCR in the absence of Triazole shows only generation ofunspecific side products whereas the addition of either concentration ofTriazole significantly improves enzyme activity generating the desiredPCR product even under fast cycling PCR condition.

Example 3

A SYBR Green I based PCR reaction was conducted in the presence ofunmodified Taq DNA Polymerase (QIAGEN) serving as a control reaction andHotStarTaq DNA polymerase (QIAGEN). Each reaction (20 μL reactionvolume) contained QuantiTect SYBR Green PCR Master Mix withoutHotStarTAQ DNA Polymerase and without (NH₄)₂SO₄. 1.0 units of therespective DNA Polymerase and 0.5 μM primer A: 5′-GTC ACC TTC ACC GTTCCA GT-3′ (SEQ ID NO:5), 0.5 μM primer B: 5′-CTC TTC CAG CCT TCC TTCCT-3′ (SEQ ID NO:6), PCR grade water and in case of testingammonia-containing compounds the different ammonium compounds (forammonia compound and concentration see table). Each reaction contained10 ng human genomic DNA as amplification substrate. PCR reactions werestarted with an initial enzyme activation step at 95° C. for 10 minutes(witch is 5 minutes shorter than the time described in the QIAGENQuantiTect SYBR Green protocol) followed by 40 cycles: 94° C. for 15seconds and 55° C. for 39 seconds and 72° C. for 40 seconds. AverageC_(T) values were determined from triplicate reactions comparingreactions without the addition of ammonia compounds and with ammoniacompounds in different concentrations (for ammonia compound andconcentration see FIG. 2).

Result: While the addition of ammonia compounds showed no effect (orinhibition at high concentrations) on the enzyme activity when usingunmodified Taq DNA polymerase, reactions containing ammonia compoundsshowed clearly higher enzyme activity when using a chemically modifiedenzyme like HotStarTaq DNA Polymerase.

Example 4

A fast PCR reaction with significantly shortened cycling times wasconducted containing the chemically modified HotStarTaq DNA Polymerasein the absence (control reaction; 0 mM ammonium chloride) and presenceof increasing ammonium chloride concentrations (10, 20, and 30 mM in thefinal reaction mixture). Each PCR reaction contained 1.5 unitsHotStarTaq DNA Polymerase, 10 ng template human or mouse genomic DNA,200 μM of each dNTP, 0.5 μM of each primer for each different PCR assay(Assay A, primer A1: 5′-GCT TGA GCA ACC TGG CTA AGA TAG AGG-3′ (SEQ IDNO:7), primer A2: 5′-GAG TTA GCA GGA GGC TGG ATG CAG ATG-3′ (SEQ IDNO:8); Assay B, primer B1: 5′-CCA CAA TGG ACA TCA CAC AAG TGA G-3′ (SEQID NO:9), primer B2: 5′-GAT CTT TCT GCC CAG ATA CCA TTC G-3′ (SEQ IDNO:10); Assay C, primer C1: 5′-CCT TGC CTT AGA TGT GTC GGC A-3′ (SEQ IDNO:11), primer C2: 5′-CCC AAA CCC AAC CCA TAC ACA C-3′ (SEQ ID NO:12))and PCR grade water. PCR reactions were started with an initial enzymeactivation step at 95° C. for 5 minutes followed by 35 cycles: 96° C.for 5 seconds, 60° C. for 5 seconds and 68° C. for 30 seconds/kb PCRproduct size. PCR products were analysed on a 1% TAE agarose gel. M: DNAsize ladder (FIG. 3).

Result: Addition of increasing amounts of ammonium chloride leads to anincrease of overall enzyme activity of the chemically inactivated enzymewhen compared to the reaction that do not contain any ammonia compound.

Example 5

In order to demonstrate the synergistic cumulative effect of acombination of an ammonia compound and an azole, a fast PCR reactionwith significantly shortened cycling times was conducted containing thechemically modified HotStarTaq DNA Polymerase in the presence of 20 mMammonium chloride in the absence (control reaction 0) and presence ofincreasing of Triazole concentrations (100, 200, 300, 400 and 500 mM inthe final reaction mixture). System C shown in Example 4 was chosen asmodel system. Each PCR reaction contained 1.5 units HotStarTaq DNAPolymerase, 10 ng template human genomic DNA, 200 μM of each dNTP, 0.5μM of each primer A: 5′-CCT TGC CTT AGA TGT GTC GGC A-3′ (SEQ ID NO:11),primer B: 5′-CCC AAA CCC AAC CCA TAC ACA C -3′ (SEQ ID NO:12), and PCRgrade water. PCR reactions were started with an initial enzymeactivation step at 95° C. for 5 minutes followed by 35 cycles: 96° C.for 5 seconds, 60° C. for 5 seconds and 68° C. for 45 seconds. PCRproducts from duplicate reactions were analysed on a 1% TAE agarose gel.M: DNA size ladder. Due to a handling error during PCR setup, thereaction indicating the assay in the presence of 200 mM Triazole showsno result (FIG. 4).

Result: Compared to the reaction containing only 20 mM ammonium chloridein the absence of Triazole, addition of Triazole shows a clear benefitfor the enzyme activity of the chemically modified enzyme. Although theeffect of Triazole starts to become less significant at very highconcentration of 500 mM, the activity is still higher than in thereaction without Triazole. This example indicates that a combination ofammonia compounds and azole can work synergistically to enhance enzymeactivity of a chemically modified enzyme. Additionally tested compoundsthat showed no improvement were C₆H₁₇N₃O₇ (ammonium citrate), H₉N₂O₄P(di-ammonium hydrogen phosphate), C₇H₉NO₂ (ammonium benzoate), (NH₄)₂CO₃(ammonium carbonate), (NH₄)₂SO₄ (ammonium sulfate).

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A method for the enhancement of the reactivationof thermostable reversibly inactivated enzymes, comprising: reactivatingat least one thermostable reversibly inactivated enzyme in the presenceof an azole.
 2. The method according to claim 1, wherein the azolecomprises a nitrogen containing 5-membered cyclic compound havingFormula 1,

wherein each X is, independent of the others, CH, CR, or N; and R is H,substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or arylgroup, a hydroxyl, amino, nitro, carboxylic acid, ester, sulfonic acid,ether, or halogen group.
 3. The method according to claim 1, wherein theazole comprises nitrogen containing 5-membered cyclic compound havingFormula 2,

wherein each X is, independent of the others, CH, CR, N, O, or S; andeach R is, independent of the others, H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, alkoxyl, or aryl group, a hydroxyl, amino,nitro, carboxylic acid, ester, sulfonic acid, ether, or halogen group.4. The method according to claim 3, wherein each X is, independent ofthe others, CH or N in any combination.
 5. The method according to claim1, wherein the azole comprises a nitrogen containing bicyclic compoundhaving Formula 3,

wherein each X is, independent of the others, CH, CR, N, O, or S; andeach R is independent of the others, H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, alkoxyl, or aryl group, a hydroxyl, amino,nitro, carboxylic acid, ester, sulfonic acid, ether, or halogen group.6. The method according to claim 1, wherein reactivating is also in thepresence of an ammonium salt.
 7. The method according to claim 1,wherein the concentration of the azole is from about 2 mM to about 1M.8. The method according to claim 1, wherein the concentration of theazole is from about 0.5 mM to about 0.5 M.
 9. A method for theenhancement of the reactivation of thermostable reversibly inactivatedenzymes, comprising: reactivating at least one thermostable reversiblyinactivated enzyme in the presence of a pyridine compound having Formula4,

wherein each X is, independent of the others, CH, CR, or N; and R is ahydroxyl, amino, thiol, or amino alkyl group.
 10. The method accordingto claim 9, wherein reactivating is also in the presence of an ammoniumsalt.
 11. The method according to claim 9, wherein the concentration ofthe pyridine compound is from about 2 mM to about 1M.
 12. The methodaccording to claim 9, wherein the concentration of the pyridine compoundis from about 0.5 mM to about 0.5 M.
 13. A method for the enhancement ofthe reactivation of thermostable reversibly inactivated enzymes,comprising: reactivating at least one thermostable reversiblyinactivated enzyme in the presence of a compound comprising anN-hydroxide having Formula 5,

wherein each R is, independent of the other, H, substituted orunsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl group, or anacyl group or derivatives thereof.
 14. The method according to claim 13,wherein reactivating is also in the presence of an ammonium salt. 15.The method according to claim 13, wherein the concentration of theN-hydroxide compound is from about 2 mM to about 1M.
 16. The methodaccording to claim 13, wherein the concentration of the N-hydroxidecompound is from about 0.5 mM to about 0.5 M.
 17. A method for theenhancement of the reactivation of thermostable reversibly inactivatedenzymes, comprising: reactivating at least one thermostable reversiblyinactivated enzyme in the presence of a compound comprising a hydrazinehaving Formula 6,

wherein each R is, independent of the other, H, substituted orunsubstituted alkyl, alkenyl, alkynyl, alkoxyl, or aryl group, or anacyl group or derivatives thereof.
 18. The method according to claim 17,wherein reactivating is also in the presence of an ammonium salt. 19.The method according to claim 17, wherein the concentration of thehydrazine compound is from about 2 mM to about 1M.
 20. The methodaccording to claim 17, wherein the concentration of the hydrazinecompound is from about 0.5 mM to about 0.5 M.
 21. A method for theenhancement of the reactivation of thermostable reversibly inactivatedenzymes, comprising: reactivating at least one thermostable reversiblyinactivated enzyme in the presence of a compound comprising an ammoniumsalt.
 22. The method according to claim 21, wherein the ammonium saltcomprises an inorganic counterion, an organic counterion, a metal oxidecounterion, or a metal complex counterion.
 23. The method according toclaim 21, wherein the concentration of the ammonium salt is from about 2mM to about 1M.
 24. The method according to claim 21, wherein theconcentration of the ammonium salt is from about 0.5 mM to about 0.5 M.25. A kit comprising at least one thermostable reversibly inactivatedenzyme and at least one of the compounds selected from the groupconsisting of an azole having Formula 1, Formula 2, or Formula 3, apyridine having Formula 4, a N-hydroxide having Formula 5, or ahydrazine having Formula 6:

wherein each X is, independent of the others, CH, CR, N, O, or S; andeach R is, independent of the others, H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, alkoxyl, or aryl, a hydroxyl, amino, nitro,carboxylic acid, ester, sulfonic acid, ether, or halogen group.